EP0335244B1 - Solid-phase analytical device and method for using same - Google Patents

Solid-phase analytical device and method for using same Download PDF

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Publication number
EP0335244B1
EP0335244B1 EP89105114A EP89105114A EP0335244B1 EP 0335244 B1 EP0335244 B1 EP 0335244B1 EP 89105114 A EP89105114 A EP 89105114A EP 89105114 A EP89105114 A EP 89105114A EP 0335244 B1 EP0335244 B1 EP 0335244B1
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Prior art keywords
control area
analyte
area
test device
assay
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German (de)
French (fr)
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EP0335244A3 (en
EP0335244A2 (en
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William E. Brown, Iii
Sarah E. Safford
John M. Clemens
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Abbott Laboratories
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Abbott Laboratories
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521Single-layer analytical elements
    • G01N33/523Single-layer analytical elements the element being adapted for a specific analyte
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/528Atypical element structures, e.g. gloves, rods, tampons, toilet paper
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54386Analytical elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761Hepatitis B
    • G01N33/5764Hepatitis B surface antigen
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/805Test papers
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/81Packaged device or kit
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/807Apparatus included in process claim, e.g. physical support structures
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/807Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808Automated or kit

Description

  • This invention relates generally to analytical devices having a plurality of readable results through the use of procedural control and analyte binding areas on a single reaction site. More particularly, the present invention relates to novel devices useful in the performance of binding assays, and to improved analytical devices. The concepts of the present invention are especially advantageous in the performance of enzyme immunoassay of biological fluids and products such as serum, plasma, whole blood, urine, spinal and amniotic fluids, mucus and the like.
  • Background Art
  • Various analytical procedures and devices are commonly employed in assays to determine the presence and/or concentration of substances of interest or clinical significance which may be present in fluids or other materials. Such clinically significant or interesting substances are commonly termed "analytes", and can include, for example, antibodies, antigens and the broad category of substances commonly known by the term "ligands". Particularly with respect to the diagnosis and treatment of disease or other conditions of the human body, the accurate determination, on a timely basis, of the presence or amount in biological fluids of certain analytes which are of clinical significance can have a profound influence on the ability of health care professionals to treat and manage pathological physical disorders, or to make an early and accurate determination of physiological conditions such as pregnancy.
  • One assay methodology which has been increasingly applied in the diagnosis of various disorders and conditions of the human body is the binding assay, and in particular the type of binding assay known as enzyme immunoassay (EIA). EIA techniques take advantage of the mechanisms of the immune systems of higher organisms, wherein antibodies are produced in response to the presence of substances (i.e., antigens) in the organisms which are pathogenic or foreign to the organisms. One or more antibodies are produced in response to and are capable of reacting with a particular antigen, thereby creating a highly specific reaction mechanism which can be advantageously utilized, in vitro, to determine that particular antigen.
  • Conventional EIA procedures involve a series of wet chemistry steps using liquid reagents, wherein an analyte in a sample biological fluid under assay, e.g., an antigen or antibody in a test sample of urine, whole blood or serum, is detected. In one type of EIA procedure, the analyte in the sample initially becomes bound to a corresponding antigen or antibody reagent which is introduced into the sample. Then, another antigen or antibody is introduced. This second antigen or antibody, however, is one which has been labeled or conjugated with an enzyme or other substance capable of producing or causing, often when reacted with or in the presence of an additional, suitable indicator reagent such as a chromogen or dye, a detectable response such as color development. The detectable response so produced can then be read and interpreted, visually or instrumentally, as an indication or measure of the presence or amount of the antigen or antibody present in the original sample.
  • Solid-phase EIA procedures are generally considered preferable for both antibody and antigen assays because of their safety, ease of use, specificity and sensitivity by comparison with heretofore-employed liquid reagent binding assay techniques such as radioimmunoassay (RIA), and other conventional wet chemistry methodologies. Moreover, the possibility of reading color development instrumentally, such as by use of a spectrophotometer, is a feature of many solid-phase EIA techniques which has resulted in their wide-spread use.
  • Thus, in one type of conventional solid-phase EIA "sandwich" assay, a test sample suspected of containing an antibody or antigen of interest is initially contacted by a solid, substantially inert plastic or glass bead or other support material which has been previously coated with a protein or another substance capable of reaction with the antigen or antibody to retain it on the surface of the support, either by immobilization of the antigen or antibody on the surface or by chemical binding therewith. A second antigen or antibody, which is usually conjugated (linked chemically) with an enzyme, is then added and this second species becomes bound to its corresponding antibody or antigen on the support. Following one or more washing step(s) to remove unbound material, an indicator substance, for example, a chromogenic substance reactive in the presence of the enzyme, is then added and, because of its sensitivity to the presence of the enzyme, produces a detectable color response. The development of the color response, its intensity, etc. can be determined visually or instrumentally, and correlated with the amount of antigen or antibody which was present in the sample.
  • Such assay techniques, and the use of the solid-phase bead or other types of supports for conducting the immunological reactions and changes necessary in such assays, are well known, but have not been without drawbacks. For example, the necessity of elaborate apparatus for conducting the assay and for containing the liquid reagents employed often results in substantial labor and equipment costs, especially for low-volume testing of individual samples. Moreover, the accuracy and reproducibility of such assays may often be less than optimum, since it is sometimes difficult to manufacture conventionally-coated solid supports and other apparatus associated with such assays so that, for a particular assay, all of the materials used therein are specifically designed to meet predetermined sensitivity and specificity requirements. Accordingly, a need exists for relatively simple, easy-to-use and comparatively inexpensive solid-phase materials and analytical devices which advantageously can be used in EIA procedures, and which are capable of producing rapid, sensitive and highly reproducible results comparable to conventional methodologies such as the aforedescribed, without the necessity for numerous, cumbersome wet chemical steps or complex instrumentation.
  • EP-A-217 403 (Abbott) describes a solid phase immunoassay device having a reaction surface onto which reagents and sample are applied. Beneath the reaction surface is an absorbent member designed to facilitate the flow of sample and reagents through the reaction surface and into the absorbent member. The reaction surface contains specific control areas, including a positive control area, a negative control area and an analyte binding area. One method of forming such control areas is by the immobilization of microparticles within a fibrous matrix, the microparticles bearing specific binding agents. The flow of fluid in this device is in a direction transverse to the plane of the reaction surface.
  • EP-A-269 876 (Abbott) discloses a funneling or focusing device to be used in conjunction with the above-described assay device. The disclosure teaches that the lower opening of the funnel device should correspond approximately to the dimensions and shape of the positive control area and the analyte binding area.
  • EP-A-299 359 (Abbott) describes another solid phase immunoassay device. This device is also a flow-through apparatus in the sense that fluid flow is generally transverse to the plane of the reaction surface. This device differs in its simpler and less expensive construction from the device of EP-A-217 403 described above.
  • EP-A-264 036 (Abbott) relates to the discovery that Chlamydial antigens can be captured directly from a sample on a support consisting of polytetrafluoroethylene (PTFE or teflon). In a particularly preferred embodiment, the PTFE support is configured as microparticles which can be imbedded in the fibrous matrix to comprise an analyte binding area in the device of EP-A-217 403.
  • SUMMARY OF THE INVENTION
  • The present invention directly addresses the foregoing need, and provides, in one aspect, a novel device useful in the performance of a binding assay to determine the presence or amount of an analyte in a test sample, and an assay utilizing the device. In another aspect, the present invention provides an improved, solid-phase analytical device, and a binding assay using the device, which is highly advantageous over devices and assay methods of the prior art. In yet another aspect, the device of the present invention is a chromatographic test strip device containing on-board procedural controls.
  • An improved device of the invention comprises a reaction site for performing a binding assay.
    The concepts of the invention are advantageous not only in the performance of binding assays to determine the unknown presence or concentration of various analytes in test samples, but also to provide on-board controls for solid-phase assay devices. As described in more detail, infra, the preferred solid-phase analytical devices in accordance with the invention incorporate assay controls, such as a visible positive control area for displaying a negative result which enables unambiguous interpretation of test results in a visual assay system. Also, for example, a preferred procedural control device utilizing the concepts of the invention can comprise a material having within its porous matrix of fibers a substance capable of producing a detectable response to an analyte in a test sample under analysis.
  • According to the present invention improved methods for performing a binding assay, utilizing the device of the invention, are provided. In one such preferred method, a sample containing an analyte, e.g., antigen or antibody, is contacted with a reaction surface made from the a porous material which can comprise solid particles returned and immobilized on the material. The method of contacting the reaction site is by means of chromatographic flow where the reaction site is located on a porous chromatographic strip. The analyte becomes bound to the reagent upon the particles retained within the material the reaction surface is then contacted with a second "labeled" reagent also capable of becoming bound to the analyte which is bound by the reagent retained within the material. Alternatively, the second reagent can be an unlabeled antibody, followed then by addition of labeled substance or reagent directed against the antibody (Amplification or Indirect immunoassay). Thereafter, unbound material is removed, e.g., by washing, and the device is contacted with an indicator substance which, in the presence of the "label" of the second reagent, produces a detectable response which is indicative of the presence and/or amount of the analyte in the sample. Such a detectable response can be read visually or instrumentally, and can advantageously be a color response, most desirably in the form of the visible appearance of a "+" or "-" sign to indicate the result of the assay, particularly if only positive or negative results, respectively, from the assay are necessary or desired. Alternatively, quantitative or semi-quantitative results can be obtained by visually or instrumentally reading the detectable response.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • Figs. 1A, 1B and 1C are top plan views of a particularly preferred embodiment of a chromatographic device.
  • Figs. 2A, 2B and 2C are top plan views of an alternate embodiment of a chromatographic device.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The novel devices of the present invention, containing readable responses, although applicable to many types of analysis, are especially advantageous when used in immunoassays, to improve conventional solid-phase immunoassay techniques for performing colorimetric or other EIA of biological fluids, such as previously described. Moreover, devices produced in accordance with the invention are relatively easy to use, and require fewer procedural steps and less complex assay technique, by comparison with prior art assays, and also provide the additional advantage of rapid quantitative, semi-quantitative or- qualitative results for testing of unknown samples. The material and devices are additionally adapted for advantageous use as controls, e.g., to assess the accuracy and reliability of such assays. Moreover, during manufacture, devices of the invention can be relatively easily made. Assays utilizing such devices of the invention have also been found to be highly sensitive to various levels of analytes. The foregoing advantages, as well as other advantages, will be apparent from the detailed description of the invention as set forth herein.
  • The concepts of the present invention are applicable to various types of binding assays. Schematic representations of examples of several such types of assays for antigen and antibody analytes can be set forth as follows. However, it will be appreciated that one skilled in the art can conceive of many other types of assays, including analytes other than antigens or antibodies, to which the present inventive concepts can be applied.
    • 1. Direct Assays
      • A. Antigen (Ag) Assay
        Figure imgb0001

        Ab, may or may not be the same as Ab₂ and may consist of a variety of monoclonal antibodies or polyclonal antibodies.
        Examples of antigen analytes determinable according to the invention using the foregoing reaction scheme include, without limitation, Strep-A, beta-hCG and hepatitis B surface antigen (HBsAg).
      • B. Antibody (Ab) Assay
        • i)
          Figure imgb0002

          Analyte examples (not limitative):
             a-HTLV-III;
             a-HBc-IgM;
             a-Rubella
        • ii)
          Figure imgb0003

        Analyte example: a-HAV-IgM
    • 2. Indirect Assays
      Antigen Assay
      Figure imgb0004

      This is a group of assays where the label is not directed against the analyte. In this embodiment, anti-Ab, may be directed against Ab, in general, or may be directed against one or more functional groups incorporated into Ab.
      It is also desirable, in some cases, to capture the analyte directly on the solid phase, as follows:
      Figure imgb0005
    • 3. Competitive Assays
      Antibody Assay
      Figure imgb0006

      In assay scheme 3, both the sample and the label are directed against the antigen on the solid phase.

    The amount of label bound reflects the amount of antibody in the sample.
  • It is to be appreciated that in addition to the capability of the device 11, as described infra, to produce a visually-readable response such as color development indicative of an analyte in a test sample, instrumental determination can be made of a detectable response therefrom, e.g., corresponding to the reflectance of visible light, or intensity of fluorescence or the like, produced by the matrix 12 as a result of the chemical and biological reactions and changes which occur therein when an assay is performed. Accordingly, the detectable response from the device 11 can be measured by, for example, a conventional spectrophotometer. For example, if the detectable response in the matrix 12 produced by the reactions and changes during a particular assay is one wherein a color is developed, and wherein increasing color development indicates an increasing level of a particular analyte in a test sample undergoing analysis, then a diminishing level of light reflected from the matrix 12 to the spectrophotometer corresponds to that increased level of analyte in the sample. The interpretation of such results is capable of being accomplished in ways well known to those skilled in the art, such as by conversion of analog signals generated by the detector of the spectrophotometer to digital information using largely conventional electronics. Such electronics are also well known to those skilled in the art, and are capable of producing a human-readable signal from such digital information which corresponds or correlates to the presence and/or amount of analyte in the test sample.
  • In accordance with the invention, the material useful in the analytical device and methods of the invention comprises a porous, fiber matrix. By "porous" is meant that the matrix is composed of a material into which fluids can flow and can easily pass through. In the material useful in the present invention, the property of porosity can be achieved simply by selection of an appropriate raw material, such as glass, cellulose, plastic nylon or other fibrous material well known to those skilled in the art.
  • For example, an especially preferred material for use is "Whatman GF/D" glass fiber filter paper, which has a nominal thickness of 0.8 mm (0.032 inch). The thickness of such a material is not critical, and will be a matter of choice for the routineer, largely based upon the properties of the sample (and analyte) being assayed, such as its fluidity and the necessity to retain enough of the sample within the material for a long enough time to enable sufficient binding of the analyte.
  • In addition, the fibrous material preferably has a plurality of substantially spherical, solid particles having an average diameter of from 0.1 to 10 µm or more, most preferably from 0.1 to 5 µm retained and immobilized upon the fibers of the material. By "retained and immobilized" is meant that the particles, once upon the fibers of the material, are not capable of substantial movement to positions elsewhere within the material, (i.e., to other fibers), or cannot be removed completely from the material without destruction thereof. The mechanism by which the particles are so retained and immobilized is not known, but may be due to physical surface attractions between the fibers and the particles, and/or between the particles themselves. The particles can be selected by one skilled in the art from any suitable type of particulate material known generally as "microparticles"; such particles are typically composed, e.g., of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate or similar materials. Whatever type of microparticles is selected for use in the invention, it is important that the substance or substances of which the particles are composed be capable of holding on the surface of the particles a substance capable of reaction with an analyte in a test sample, e.g., antibody or antigen, or a combination thereof, or be itself capable of holding an analyte on the surface of the particles. Moreover, the size of the particles is not critical, and so long as the average diameter of the particles is substantially within the aforestated range (although it is preferred that the average diameter of the particles be smaller than the average pore size of the fibrous matrix), any type of particles having the foregoing properties is suitable for use.
  • The analytical devices provided by the invention, it is to be appreciated, can be advantageously employed in a wide variety of otherwise well-known assay techniques and procedures, and are not limited in application to the specific immunoassay techniques described in detail herein. They can thus be used in so-called "competitive binding" assays or similar binding assay procedures, and in addition, can be employed in other assays such as typical enzyme assays for such analytes as glucose, uric acid or the like, which are not immunoassays but which can advantageously be carried out by initially retaining at least one reagent used in such assays upon the particles within the material or reaction matrix of a device of the invention. It will be readily apparent to those skilled in the analytical arts that the instant invention can be profitably applied to a wide variety of uses in various types of assay procedures, and thus is in no way limited to the specific details of the assays and procedures described herein.
  • The novel analytical devices, produced in accordance with the principles of the instant invention can, however, be especially advantageously employed in enzyme immunoassays, particularly so-called "sandwich" and indirect enzyme immunoassays. Such assays can be performed using the material and devices of the invention in a manner which is substantially more simple than typical "bead" or other assays of the prior art which require relatively elaborate, time-consuming and costly equipment and materials. Such assays also have been found to be capable of surprising sensitivity. A generalized example for one presently preferred "sandwich" immunoassay procedure utilizing the material of the device of the instant invention is as follows:
    • Step a) Retention of antibody or antigen upon the particles in the material, forming a reaction matrix, as previously described;
    • Step b) Application of a test sample containing antigen or antibody to be determined to the matrix;
    • Step c) Application of an enzyme-conjugated antibody or antigen to the antigen or antibody of Step b);
    • Step d) Washing, to remove unbound material; and
    • Step e) Application of an indicator substance which, in the presence of the enzyme portion of the conjugate of Step c), produces a detectable color or other response in the reaction matrix.
  • A more detailed discussion of how such "sandwich" assay procedures can advantageously be carried out using the device of the present invention is set forth in the Examples, infra.
  • In accordance with the present invention, a detectable response is produced at the reaction surface or site on a porous material or reaction matrix of an analytical device; the response is one which is indicative of the presence and/or amount of an analyte in a sample under analysis. Such a detectable response, in preferred embodiments of the invention, can be color development following a series of assay steps, such as those previously described, or can be any number of responses well known in the analytical arts and used for similar purposes. For example, the response produced can be one of fluorescence, provided appropriate reagents are employed in the assay, as is well known to those skilled in the art. The response can be also chemiluminescence, or any of a variety of radiative energy responses (e.g., radioactive emissions) detectable either visually, or instrumentally by various known equipment. Thus, it is to be especially appreciated that in use of the devices of the invention, many different types of detectable responses are possible and desirable and the inventive concepts are not limited thereby.
  • "On-board" procedural control areas are provided on solid phase analytical devices to simultaneously display detectable responses corresponding to a positive control (which will display a detectable response indicative of a valid assay result, regardless of the presence or absence of an analyte of interest in a test sample), a negative control (which will display a detectable response change only if the assay results are invalid) and the sample analyte in a single analytical device reaction site. Typical analytical devices for use with the present invention can include a test strip for chromatographic assay devices (e.g., paper) or thin layer chromatographic assay devices (e.g., nitrocellulose) devices in which one or all the reagents are contained in separate zones of a single strip or other porous material in communication therewith.
  • The same volume of a test sample and assay reagents are simultaneously placed in contact with the procedural controls and test areas, thereby avoiding the necessity of separate control tests as generally practiced in the art. The method of application of sample and reagents to the reaction site can be any manner appropriate for the particular device used.
  • In the chromatographic assay device or test strip of the invention, the sample and reagents are applied to the strip and allowed to flow to and through the reaction site containing the procedural controls and test areas. Regardless of the particular device used, the reaction site containing the procedural controls (negative control and positive control) and analyte binding area is simultaneously contacted by applications of reagent and sample.
  • It will be apparent to those skilled in the art that the procedural controls or readable results in the device of the invention may be similarly employed with any analytical device having a reaction site capable of simultaneously displaying a plurality or multiplicity of reaction results. Such other types of reaction surfaces include, for example, coated or uncoated fiber matrices, filters, paper or membranes, relatively planar solid surfaces and the like.
  • Referring now to Figures 1A-C and 2A-C, on-board negative and positive control areas 30 and 32, respectively, are preferably provided at the reaction site on the reaction surface or matrix 12 of the analytical device 11. Device 11 is a chromatographic strip. The negative and positive control areas may function in a quantitative manner thereby functioning as negative and positive assay reference controls, or may function in a qualitative manner thereby functioning as procedural controls indicating the validity of procedures and reagents used in the performance of an assay. As used herein, the term "control" includes both quantitative and qualitative embodiments. Negative control area 30 is formed by maintaining the control area 30 of the matrix 12 free of substances which will retain the enzyme label or other signal response material during the normal use of the device 11 in the performance of a binding assay, as described herein.
  • Positive control area 32 is formed by providing a substance capable of binding the enzyme label or other signal response material within the control area 32 of the matrix, regardless of the presence or absence of the analyte of interest in a test sample. As used in connection with the particularly preferred reaction matrix as previously described, positive control area 32 may be formed by coating the microparticles within the control area 32 with the analyte, or other substances capable of binding or retaining the enzyme label within the area 32 during performance of a binding assay. In addition, one or more analyte binding area(s) 34 are provided on the matrix 12 for binding or retaining the analyte of interest from a test sample on the area 34 during the performance of a binding assay. The analyte binding area(s) 34 may be formed in the particularly preferred reaction matrix material described herein by coating the microparticles within the area(s) 34 of the matrix 12 with a substance, such as antigen or antibody, capable of binding the analyte.
  • The positive control area 32 and the analyte binding area(s) 34 may be provided in any configuration which facilitates ease of use of the device 11 in the performance of a binding assay. However, it is presently preferred to provide the positive control area and the analyte binding area in an interactive configuration in which the positive control area interacts with the analyte binding area upon the occurrence of a positive test result to form a first representational symbol having a known meaning to the user, and the positive control area acts alone upon the occurrence of a negative test result to form a second representational symbol having a known meaning to the user different from that of the first representation symbol. Interactive positive control and analyte binding areas are best shown in the particularly preferred embodiment of Figures 1A-C, wherein the positive control area 32 is formed in the shape of a rectangular bar or "-" sign, while the analyte binding areas 34 are formed in the shape of rectangular bars on opposite sides of, and oriented perpendicularly with respect to, the positive control area 32. Accordingly, in use of the devices of Figures 1A-C, a positive test result obtained from the proper use of the device 11 will result in a detectable response, in the shape of a "+" sign, in both the positive control area 32 and the analyte binding areas 34, as shown in Figure 1C, indicating a "+" or positive test result to the user. A negative test result obtained from the proper use of the devices will result in a detectable response, in the shape of a "-" sign, in only the positive control area 32, as shown in Figure 1B, indicating a "-" or negative test result to the user. If the binding assay is improperly conducted, or if reagents used in the assay function improperly, no detectable response is obtained in either the positive control area 32 or the analyte binding areas 34, as shown in Figure 1A, indicating an invalid test result. In addition, any detectable response in the negative control area 30, such as may be caused by non-specific binding or failure to properly perform washing steps in the performance of the assay, may be indicative of an invalid test result. The configuration of Figures 1A-C is presently particularly preferred since it provides immediate information to the user in unambiguous, symbolic form as to the positive (+) or negative (-) nature of the test result, and as to the validity of the assay.
  • Alternatively, the procedural control areas and the analyte binding areas may be provided in other configurations, as desired. In the alternate embodiment of Figures 2A-C, the positive control area 32 and the analyte binding area 34 are formed in the shape of dots, as shown. Thus, a positive test result is indicated by the presence of two dot-shaped detectable response areas, as shown in Figure 2C, a negative test result is indicated by the presence of a detectable response only in the positive control area 32, as shown in Figure 2B, and invalid test result is indicated by the lack of a detectable response as shown in Figure 2A. Other equivalent configurations for the negative control area 30, the positive control area 32 and the analyte binding area(s) 34, such as other symbols, numbers and the like, will be readily apparent to those skilled in the art.
  • EXAMPLES
  • The following Examples illustrate the preparation of a solid-phase reaction matrix containing microparticles which can be used in the device of the present invention. Illustration of readable results through the use of procedural control and analyte binding areas on a single reaction site can be found in EP-A-0 217 403 in reference to a flow-through device in which however, in a manner quite different from what occurs in the device of the present invention, the flow of fluid sample is in a direction transverse to the plane of the reaction surface. The Examples are intended to be only illustrative, and in no way to be construed as placing limitations upon the scope of the invention, which scope is defined solely by the appended claims.
  • Unless otherwise indicated, all percentages expressed herein are by weight.
  • Example 1: Preparation of Antibody-Coated Microparticles
  • 100 microliters of carboxylate-modified microparticles (2.5% solids; 0.45 µm average diameter; commercially available from Polyscience and Seragen) were added to 1.0.milliliters (ml) of methyl ethyl sulfonate (MES) buffer (5 millimolar (mM), pH 4.75) and 75 microliters of antibody solution (beta-hCG) (2 milligrams per milliliter (mg/ml)). The solution was stirred and then 100 ml of 1-Ethyl-3(3-Dimethylaminopropyl) carbodimide HCl (EDAC) (2 mg per 10 ml H₂0) were added. The solution was stirred overnight at 2-8 degrees C, after which the microparticles were isolated by centrifugation, washed twice with 0.1% "Tween®-20" solution, and resuspended in "PBS" Phosphate Buffered Saline (0.01 M KH₂PO₄; 0.15M NaCl: pH 7.2) to yield a 0.125% solution. After resuspension in PBS, the particles were stored at 2-8 degrees C, for subsequent use in the following procedures.
  • Example 2: Preparation of Solid-Phase Reaction Matrix
  • 50 microliters of the antibody-coated microparticles from Example 1 were added dropwise to the center of a Whatman GF/D glass filter; 100 microliters of pig sera were then added and the filter and microparticles incubated for 30 minutes in a humidity chamber at room temperature. After this time, the filter, now containing the microparticles, was washed three times in 300 microliters of PBS buffer. The filter was then stored in a humidity chamber until it was used in an immunoassay. The microparticles were observed, by scanning electron microscopy, to have been irreversibly trapped or agglomerated on the glass fibers of the filter material.
  • It is to be noted that, in addition to the techniques described in the foregoing Example, antibody (or antigen) may be attached to the particles by a variety of methods; e.g., adsorption or use of various chemical activators. Also, it is to be appreciated that the particles can be added to the fibrous matrix after, for example, animal sera has been added, and that the use of such sera is not of critical importance. Therefore, the order of addition of the particles to the matrix and treatment thereof after or before incorporation into the matrix is not critical to the present invention. Moreover, it will be appreciated that coated fibrous materials, such as polystyrene-coated glass, can be used in place of the glass filter matrix material specifically described herein, and the advantages of the invention can also be realized thereby.
  • Example 3: Analytical Device Incorporating Procedural Control Area
  • The following is a general example of the preparation of an analytical device according to the invention, which additionally incorporates a procedural control area for determining non-specific reactivity (interference) of the sample with the solid phase.
  • Reaction matrices utilizing the material of the invention can be prepared substantially as previously described, and the particles incorporated into the material in a pattern having substantially the overall shape of a "cross". The vertical axis of the "cross" can be formed of the particles having an analyte-binding substance upon their surfaces, whereas the horizontal axis of the "cross" can be formed of a substance capable of binding the enzyme label (i.e., antibody capable of becoming "conjugated" or attached to the label). Accordingly, when these reaction matrices are used in an assay e.g., for beta-hCG, if no detectable level of analyte is present in the sample only the "procedural control area" of the matrix will produce a detectable response, i.e., the horizontal axis of the "cross" (a "minus" sign) will develop color or another response, indicating a negative result. However, if a detectable level-of analyte is present, then the analyte will bind, along with the label, to the particles both in the horizontal and vertical axes, producing a detectable response in both axes (a "plus" sign).
  • Alternatively, the areas of the matrix in which the responses are produced can take the form of "dots", circles, numbers and the like. Thus, the microparticles can be sprayed or otherwise dispensed into the material of the matrix and incorporated therein, as previously described, in various patterns as desired. While the foregoing controls have been described in this Example as used in connection with the presently preferred matrix material of the invention, the on-board controls may be similarly employed in connection with other solid-phase analytical devices, as previously described. The advantages of incorporation of such a procedural control into the material and device heretofore described, as well as into solid phase assay devices using other types of matrix materials, include a) a control provides a measure of validation of materials for each assay run; b) a control with each assay run enables comparative interpretation of results, especially when specific patterns, such as "plus" ("+") and "minus" ("-") signs are used; and c) the incorporation of a control into each assay device provides expedient validation of the assay, allowing the user to be more confident of the assay results.
  • It is to be appreciated that various modifications and changes can be made in the specific, preferred embodiments of the invention as described in detail herein, without departing from the spirit and scope of the invention, as set forth in the following claims.
  • Where technical features mentioned in any claim are followed by reference signs, those reference signs have been included for the sole purpose of increasing the intelligibility of the claims and accordingly such reference signs do not have any limiting effect on the scope of each element identified by way of example by such reference signs.

Claims (9)

  1. A test device for use in a binding assay to determine the presence or amount of an analyte in a fluid sample upon the addition of sample, suitable reagents and label to a reaction site, wherein said reaction site has a first control area (30), a second control area (32) including means for displaying a detectable response indicative of a valid assay result, and an analyte binding area (34) including means for displaying a detectable result indicative of the presence or amount of the analyte in the fluid sample; characterized in that
       said device is a porous chromatographic strip (11) having a contact site distinct from said reaction site for contacting a fluid sample and means (12) for transporting the fluid sample by capillarity or wicking to the reaction site, such that said fluid sample flows across said reaction site from edge to edge; and in that the detectable symbol produced by the second control area (32) interacts with the detectable symbol produced by the analyte binding area (34) in the presence of analyte to form an interactive symbol representative of a positive result, while the symbol produced by the second control area (32) taken alone is representative of a negative result.
  2. A test device according to Claim 1 wherein neither the first nor the second symbol is formed if the test is performed improperly.
  3. A test device according to Claim 1 or 2 wherein the second control area (32) is configured in the shape of a rectangular bar whereby the detectable symbol produced from the second control area (32) is a minus sign ("-").
  4. A test device according to Claim 3 wherein the analyte binding area (34) is configured in the shape of two colinear rectangular half bars oriented on both sides of and substantially orthogonal to the second control area (32).
  5. A test device according to Claim 4 wherein the two colinear half bars appear to intersect the second control area (32) near its mid-point such that the symbol produced by the analyte binding area (34) taken together with the "minus" symbol produced by the second control area (32) produces a plus sign ("+").
  6. A test device according to any of Claims 1-5 wherein the label comprises a conjugate of anti-analyte antibody and a detectable moiety.
  7. A test device according to any of Claims 1-5 wherein a single addition of fluid sample or reagent transported through the porous strip contacts each area of said reaction site substantially at the same time.
  8. A test device according to any of Claims 1-5 or Claim 7 wherein the first control area (30) surrounds the second control area (32) and the analyte binding area (34).
  9. A test device according to any of Claims 1-5 wherein the first control area (30) comprises a negative control area and the second control area (32) comprises a positive control area.
EP89105114A 1986-02-18 1989-03-22 Solid-phase analytical device and method for using same Expired - Lifetime EP0335244B1 (en)

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US173979 1988-03-28
US07/173,979 US4916056A (en) 1986-02-18 1988-03-28 Solid-phase analytical device and method for using same

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US4916056A (en) 1990-04-10
CA1332807C (en) 1994-11-01
AU618586B2 (en) 1992-01-02
KR890015016A (en) 1989-10-28
DE68920176T2 (en) 1995-05-11
JPH01299464A (en) 1989-12-04
JP2818191B2 (en) 1998-10-30
KR920009420B1 (en) 1992-10-16
EP0335244A2 (en) 1989-10-04
DE68920176D1 (en) 1995-02-09
AT116443T (en) 1995-01-15
ES2068844T3 (en) 1995-05-01
EP0335244A3 (en) 1990-05-30
AU3156489A (en) 1989-09-28

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